Global Analysis of the Human RNA Degradome. Profiling the transcriptome with microarrays or sequencing approaches has become a mainstay of basic and applied biomedical research. In 2005, the deep sequencing of small RNAs was pioneered in the PI's lab, an approach that is now routine for examining miRNA populations in eukaryotic systems. The finding that miRNA profiling can be more effective than mRNA profiling for typing certain tumors emphasizes that profiling other major components of the transcriptome can lead to new and more potent disease associations. One component of the transcriptome that has been largely ignored is the population of RNA decay products, i.e., the RNA degradome. The long-term goal of this project is to facilitate the use of RNA degradome information to solve biological problems and identify new markers of human disease. To accomplish this, it is essential to demonstrate that the RNA degradome can be analyzed on a genome-wide scale and provide important information. This project seeks to achieve this for the human degradome, and test the hypothesis that regulation of RNA decay is more widespread than previously thought. An approach called Parallel Analysis of RNA Ends (PARE) will be applied to the problem. Like the first miRNA profiling studies, PARE was developed using Arabidopsis plants, in which miRNAs typically guide the cleavage of their mRNA targets, similar to what happens during RNAi in mammals. By examining the population of RNA decay products corresponding to these events (polyadenylated RNAs with a 5'monophosphate), nearly all the validated and many new miRNA targets were identified. Although this approach provides an excellent way to identify potentially important targets of miRNAs or other endogenous small RNAs that cause cleavage in human, these cases are thought to be rare. It is the use of PARE to examine the patterns of decay for each transcript in the transcriptome that is expected to have the greatest impact. Under this project, PARE libraries will be made and deeply sequenced from different ENCODE lines, knockdown mutants, and different types of decay products. Decay plots for each annotated human cDNA for each library will be generated and compared. Transcripts that exhibit altered degradation in individual or multiple samples will be identified and validated. This work will add a new dimension to existing gene expression data, and contribute significantly to it. Moreover, it will encourage investigators to examine the associations of changes in the RNA degradome with a range of human diseases. PUBLIC HEALTH RELEVANCE: This work will add a new dimension to existing gene expression data, and contribute significantly to it. Moreover, it will encourage investigators to examine the associations of changes in the RNA degradome with a range of human diseases.